Complete suspension culture of human induced pluripotent stem cells supplemented with suppressors of spontaneous differentiation.

Human induced pluripotent stem cells (hiPSCs) are promising resources for producing various types of tissues in regenerative medicine; however, the improvement in a scalable culture system that can precisely control the cellular status of hiPSCs is needed. Utilizing suspension culture without microcarriers or special materials allows for massive production, automation, cost-effectiveness, and safety assurance in industrialized regenerative medicine. Here, we found that hiPSCs cultured in suspension conditions with continuous agitation without microcarriers or extracellular matrix components were more prone to spontaneous differentiation than those cultured in conventional adherent conditions.

On the mechanisms of lysis triggered by perturbations of bacterial cell wall biosynthesis.

Inhibition of bacterial cell wall synthesis by antibiotics such as β-lactams is thought to cause explosive lysis through loss of cell wall integrity. However, recent studies on a wide range of bacteria have suggested that these antibiotics also perturb central carbon metabolism, contributing to death via oxidative damage. Here, we genetically dissect this connection in Bacillus subtilis perturbed for cell wall synthesis, and identify key enzymatic steps in upstream and downstream pathways that stimulate the generation of reactive oxygen species through cellular respiration.

Dissecting the roles of peptidoglycan synthetic and autolytic activities in the walled to L-form bacterial transition.

Bacterial cells are surrounded by a peptidoglycan (PG) wall, which is a crucial target for antibiotics. It is well known that treatment with cell wall-active antibiotics occasionally converts bacteria to a non-walled "L-form" state that requires the loss of cell wall integrity. L-forms may have an important role in antibiotic resistance and recurrent infection.

Mirubactin C rescues the lethal effect of cell wall biosynthesis mutations in .

Growth of most rod-shaped bacteria is accompanied by the insertion of new peptidoglycan into the cylindrical cell wall. This insertion, which helps maintain and determine the shape of the cell, is guided by a protein machine called the rod complex or elongasome. Although most of the proteins in this complex are essential under normal growth conditions, cell viability can be rescued, for reasons that are not understood, by the presence of a high (mM) Mg concentration.

Characterization of the L-form switch in the Gram-negative pathogen Streptobacillus moniliformis.

Almost all major classes of bacteria are surrounded by a peptidoglycan cell wall, which is a crucial target for antibiotics. It is now understood that many bacteria can tolerate loss of the cell wall provided that they are in an isotonic environment. Furthermore, in some cases the cells can continue to proliferate in a state known as the L-form.

A miniature dialysis-culture device allows high-density human-induced pluripotent stem cells expansion from growth factor accumulation.

Three-dimensional aggregate-suspension culture is a potential biomanufacturing method to produce a large number of human induced pluripotent stem cells (hiPSCs); however, the use of expensive growth factors and method-induced mechanical stress potentially result in inefficient production costs and difficulties in preserving pluripotency, respectively. Here, we developed a simple, miniaturized, dual-compartment dialysis-culture device based on a conventional membrane-culture insert with deep well plates. The device improved cell expansion up to approximately ~3.

Author Correction: Possible role of L-form switching in recurrent urinary tract infection.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Possible role of L-form switching in recurrent urinary tract infection.

Recurrent urinary tract infection (rUTI) is a major medical problem, especially in the elderly and infirm, but the nature of the reservoir of organisms responsible for survival and recolonisation after antibiotic treatment in humans is unclear. Here, we demonstrate the presence of cell-wall deficient (L-form) bacteria in fresh urine from 29 out of 30 older patients with rUTI. In urine, E.

Crucial role for central carbon metabolism in the bacterial L-form switch and killing by β-lactam antibiotics.

The peptidoglycan cell wall is an essential structure for the growth of most bacteria. However, many are capable of switching into a wall-deficient L-form state in which they are resistant to antibiotics that target cell wall synthesis under osmoprotective conditions, including host environments. L-form cells may have an important role in chronic or recurrent infections.

Lysozyme Counteracts β-Lactam Antibiotics by Promoting the Emergence of L-Form Bacteria.

β-lactam antibiotics inhibit bacterial cell wall assembly and, under classical microbiological culture conditions that are generally hypotonic, induce explosive cell death. Here, we show that under more physiological, osmoprotective conditions, for various Gram-positive bacteria, lysis is delayed or abolished, apparently because inhibition of class A penicillin-binding protein leads to a block in autolytic activity. Although these cells still then die by other mechanisms, exogenous lytic enzymes, such as lysozyme, can rescue viability by enabling the escape of cell wall-deficient "L-form" bacteria.

RodA as the missing glycosyltransferase in Bacillus subtilis and antibiotic discovery for the peptidoglycan polymerase pathway.

The bacterial cell wall is a highly conserved essential component of most bacterial groups. It is the target for our most frequently used antibiotics and provides important small molecules that trigger powerful innate immune responses. The wall is composed of glycan strands crosslinked by short peptides.

L-form bacteria, chronic diseases and the origins of life.

The peptidoglycan cell wall is widely conserved across the bacterial domain, suggesting that it appeared early in the evolution of bacteria. It is normally essential but under certain conditions wall-deficient or 'L-form' bacteria can be isolated. In Bacillus subtilis this normally requires two genetic changes.

Wall proficient E. coli capable of sustained growth in the absence of the Z-ring division machine.

The peptidoglycan cell wall is a major protective external sheath in bacteria and a key target for antibiotics(1). Peptidoglycan is present in virtually all bacteria, suggesting that it was probably present in the last bacterial common ancestor(2). Cell wall expansion is orchestrated by cytoskeletal proteins related to actin (MreB) and tubulin (FtsZ)(3).

Cell growth of wall-free L-form bacteria is limited by oxidative damage.

The peptidoglycan (PG) cell wall is a defining feature of the bacterial lineage and an important target for antibiotics, such as β-lactams and glycopeptides. Nevertheless, many bacteria are capable of switching into a cell-wall-deficient state, called the "L-form" [1-3]. These variants have been classically identified as antibiotic-resistant forms in association with a wide range of infectious diseases [4].

General principles for the formation and proliferation of a wall-free (L-form) state in bacteria.

The peptidoglycan cell wall is a defining structural feature of the bacterial kingdom. Curiously, some bacteria have the ability to switch to a wall-free or 'L-form' state. Although known for decades, the general properties of L-forms are poorly understood, largely due to the lack of systematic analysis of L-forms in the molecular biology era.

Bacterial cell morphogenesis does not require a preexisting template structure.

Morphogenesis, the development of shape or form in cells or organisms, is a fundamental but poorly understood process throughout biology. In the bacterial domain, cells have a wide range of characteristic shapes, including rods, cocci, and spirals. The cell wall, composed of a simple meshwork of long glycan strands crosslinked by short peptides (peptidoglycan, PG) and anionic cell wall polymers such as wall teichoic acids (WTAs), is the major determinant of cell shape.

Excess membrane synthesis drives a primitive mode of cell proliferation.

The peptidoglycan cell wall is a hallmark of the bacterial subkingdom. Surprisingly, many modern bacteria retain the ability to switch into a wall-free state called the L-form. L-form proliferation is remarkable in being independent of the normally essential FtsZ-based division machinery and in occurring by membrane blebbing and tubulation.

The rod to L-form transition of Bacillus subtilis is limited by a requirement for the protoplast to escape from the cell wall sacculus.

L-forms are variants of common bacteria that can grow and proliferate without a cell wall. Little is known about their molecular cell biology but they undergo a remarkable mode of proliferation that is independent of the normally essential FtsZ-dependent division machinery. We have isolated a strain of Bacillus subtilis that can quickly and quantitatively convert from the walled to the L-form state.

A widespread family of bacterial cell wall assembly proteins.

Teichoic acids and acidic capsular polysaccharides are major anionic cell wall polymers (APs) in many bacteria, with various critical cell functions, including maintenance of cell shape and structural integrity, charge and cation homeostasis, and multiple aspects of pathogenesis. We have identified the widespread LytR-Cps2A-Psr (LCP) protein family, of previously unknown function, as novel enzymes required for AP synthesis. Structural and biochemical analysis of several LCP proteins suggest that they carry out the final step of transferring APs from their lipid-linked precursor to cell wall peptidoglycan (PG).

Large ring polymers align FtsZ polymers for normal septum formation.

Cytokinesis in bacteria is initiated by polymerization of the tubulin homologue FtsZ into a circular structure at midcell, the Z-ring. This structure functions as a scaffold for all other cell division proteins. Several proteins support assembly of the Z-ring, and one such protein, SepF, is required for normal cell division in Gram-positive bacteria and cyanobacteria.

Partial functional redundancy of MreB isoforms, MreB, Mbl and MreBH, in cell morphogenesis of Bacillus subtilis.

MreB proteins are bacterial actin homologues thought to have a role in cell shape determination by positioning the cell wall synthetic machinery. Many bacteria, particularly Gram-positives, have more than one MreB isoform. Bacillus subtilis has three, MreB, Mbl and MreBH, which colocalize in a single helical structure.

The actin-like MreB cytoskeleton organizes viral DNA replication in bacteria.

Little is known about the organization or proteins involved in membrane-associated replication of prokaryotic genomes. Here we show that the actin-like MreB cytoskeleton of the distantly related bacteria Escherichia coli and Bacillus subtilis is required for efficient viral DNA replication. Detailed analyses of B.

Effects of oriC relocation on control of replication initiation in Bacillus subtilis.

In bacteria, DNA replication initiation is tightly regulated in order to coordinate chromosome replication with cell growth. In Escherichia coli, positive factors and negative regulatory mechanisms playing important roles in the strict control of DNA replication initiation have been reported. However, it remains unclear how bacterial cells recognize the right time for replication initiation during the cell cycle.

Noc protein binds to specific DNA sequences to coordinate cell division with chromosome segregation.

Coordination of chromosome segregation and cytokinesis is crucial for efficient cell proliferation. In Bacillus subtilis, the nucleoid occlusion protein Noc protects the chromosomes by associating with the chromosome and preventing cell division in its vicinity. Using protein localization, ChAP-on-Chip and bioinformatics, we have identified a consensus Noc-binding DNA sequence (NBS), and have shown that Noc is targeted to about 70 discrete regions scattered around the chromosome, though absent from a large region around the replication terminus.